Abstract

BACKGROUND:

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

RESULTS:

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

CONCLUSION:

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.

Inactivation of lin-35 or lin-15B enhances RNAi. (a) The number of genes with enhanced RNAi phenotypes in the worm strains lin-35(n745), lin-15B(n744), eri-1(mg366) and rrf-3(pk1426). The chart shows the number of genes with RNAi phenotypes that are significantly stronger in each strain than in wild-type (Bristol N2) worms. A total of 1,838 bacterial RNAi feeding strains from the Ahringer library [4] targeting 1,749 genes were tested with each worm strain. (b-f) RNAi-induced silencing of lin-35 or lin-15B enhances the dsRNA-induced silencing of a GFP transgene. Worm strain GR1401 expresses an integrated GFP transgene and a dsRNA that targets GFP mRNA for degradation, both expressed specifically in the hypodermal seam cells of the worm (arrows) [9]. (b) Control experiments used a feeding strain that does not target any C. elegans gene and causes no change in the silencing of GFP. dsRNA targeting components of the RNAi machinery such as (c) rde-4 suppress the silencing of GFP, whereas dsRNAs targeting (d) eri-1, (e) lin-35 or (f) lin-15B result in enhanced silencing of GFP. See Table 2 for quantification of this data.

lin-35 and lin-15B enhance somatic transgene silencing by an RNAi dependent mechanism. Worm strain JR667 expresses GFP specifically in the hypodermal seam cells from an integrated tandemly repeated array of the construct wIs51, which contains the scm::GFP reporter (a). Inactivation of either lin-35 (b) or lin-15B (c) results in enhanced silencing of the GFP transgene via a mechanism that is dependent upon the RNAi machinery, including dcr-1 (e-f) and rde-4 (h-i). Inactivation of dcr-1 (d) or rde-4 (g) alone results in a slight increase in GFP expression, indicating a background level of transgene silencing in wild-type worms. Worms were fed on 1:1 mixes of the indicated RNAi feeding strains and RNAi feeding experiments were performed as described in Materials and methods. Control (ctrl) RNAi experiments used the same non-targeting RNAi clone as used in Figure 1. See Table 5 for quantification of this data.

A comparison of the genetic pathways that regulate RNAi and vulval development. A subset of the synthetic Multivulva B genes, designated the synMuv B(R) genes, negatively regulate vulval induction (redundantly with the synMuv A pathway), and also negatively regulate somatic and germline RNAi. In both processes the genes mes-4 and zfp-1 act genetically downstream of, or in parallel to, the synMuv B(R) genes. The identities of the synMuv B(R) genes are given below the figure.